Chapter 11-Nucleic Acids as Therapeutic Agents Antisense RNA and oligonucleotides Ribozymes Aptamers Interfering RNAs or RNAi Gene therapy Conventional methods Genome Editing with CRISPR-Cas9 Stem cells and therapeutic cloning

Figure 11.1

Fig. 11.1 Inhibition of translation of specific RNA by antisense nucleic acid molecules Promoter antisense cDNA poly A addition signal mRNA -antisense RNA complex antisense oligonucleotide

Fig. 11.8 Ribozymes: A. Hammerhead B. Hairpin Figure 11.8

Figure 11.11 Aptamers-nucleic acid sequences (RNA or DNA) that bind tightly to proteins, amino acids or other molecules

Figure 11.13 Overview of RNA interference (RNAi)

Fig. 11.13 RNA interference (RNAi) dsRNA sense antisense Binding of dsRNA-specific nuclease Nuclease-ssRNA complex Hybridizes to mRNA cleavage mRNA is cleaved! A cellular nuclease binds to the dsRNA cleaving it into ssRNAs of 21-23 nucleotides each. The nuclease-RNA oligonucleotide complex binds and cleaves specific mRNA.

RNAi In 2006, Fire and Mello received a Nobel Prize for their RNAi work uisng Double Stranded RNA in C. elegans – see RNA Interference on YouTube: http://www.youtube.com/watch?v=UdwygnzIdVE&feature=related Discovered in petunia - see RNAi Discovered on YouTube: http://www.youtube.com/watch?v=H5udFjWDM3E&feature=related

Table 11.3

Human Gene Therapy (disease targets) AIDS Amyotrophic lateral sclerosis Cancer Cardiovasc. disease Cystic fibrosis Familial hypercholesterolemia Gaucher disease Hemophilia A Hemophilia B Hunters disease Multiple sclerosis Muscular dystrophy Rheumatoid arthritis Severe combined immunodeficiency

Human Gene Therapy Clinical Trials http://www.abedia.com/wiley/indications.php

Consider somatic vs germline gene therapy; the later is currently banned. Note that gene therapy is limited to somatic cells and disorders that are caused by a single gene.

Two types of gene therapy Ex vivo -cells are removed from the body, the gene of interest is inserted into them, the cells are cultured to increase cell numbers, and they are returned to the body by infusion or transplantation (time consuming and expensive) In vivo -a gene is introduced directly into specific cells within the body (quick and inexpensive), but targeting certain cells (e.g., bone marrow stem cells) is difficult

Vectors used to deliver genes in Human Gene Therapy Retroviruses Adenoviruses Adeno-associated viruses Herpes simplex virus Liposomes/Lipofection Naked DNA/Plasmid DNA

Severe Combined ImmunoDeficiency (SCID) See https://www.scid.net/the-scid-homepage/about-scid/ How is ADA deficiency treated? There are no real cures for ADA deficiency, but doctors have tried to restore ADA levels and improve immune system function with a variety of treatments: Bone marrow transplantation from a biological match (for example, a sibling) to provide healthy immune cells Transfusions of red blood cells (containing high levels of ADA) from a healthy donor Enzyme replacement therapy, involving repeated injections of the ADA enzyme Gene therapy - to insert synthetic DNA containing a normal ADA gene into immune cells 6-yr-old Ashanthi DeSilva-SCID sufferer treated with gene therapy-coloring at home in N Olmstead, OH (March 1993).

Cystic fibrosis transmembrane conductance regulator protein (CFTR) CFTR involved with chloride ion transport out of cells; if defective Cl- builds up inside cells and draws water inside resulting in a sticky, sugar-rich extracellular mucus.

Is gene therapy safe? What do you think? Jesse Gelsinger story Jesse Gelsinger (June 18, 1981 - September 17, 1999) was the first person publicly identified as having died in a clinical trial for gene therapy. He was 18 years old. Gelsinger suffered from ornithine transcarbamylase deficiency, an X-linked genetic disease of the liver, whose victims are unable to metabolize ammonia - a byproduct of protein breakdown. The disease is usually fatal at birth, but Gelsinger had not inherited the disease; in his case it was the result of a genetic mutation and as such was not as severe - some of his cells were normal which enabled him to survive on a restricted diet and special medications. Gelsinger joined a clinical trial run by the University of Pennsylvania that aimed to correct the mutation. On Monday, September 13 1999, Gelsinger was injected with adenoviruses carrying a corrected gene in the hope that it would manufacture the needed enzyme. He died four days later, apparently having suffered a massive immune response triggered by the use of the viral vector used to transport the gene into his cells. This led to multiple organ failure and brain death. Gelsinger died on Friday, September 17th at 2:30 PM. A Food and Drug Administration (FDA) investigation concluded that the scientists involved in the trial, including the lead researcher Dr. James M. Wilson (U Penn), broke several rules of conduct: Inclusion of Gelsinger as a substitute for another volunteer who dropped out, despite having high ammonia levels that should have led to his exclusion from the trial Failure by the university to report that two patients had experienced serious side effects from the gene therapy Failure to mention the deaths of monkeys given a similar treatment in the informed consent documentation. The University of Pennsylvania later issued a rebuttal [1], but paid the parents an undisclosed amount in settlement. The Gelsinger case was a severe setback for scientists working in the field.

Genome Editing with CRISPR-Cas9 This is Huge. https://www. youtube CRISPR-Cas9 technology allows one to edit genome sequences (delete genes, add genes, change nucleotides) https://www.youtube.com/watch?v=2pp17E4E-O8 https://www.youtube.com/watch?v=TdBAHexVYzc Clinical trials are just beginning in the US see http://www.nature.com/news/first-crispr-clinical-trial-gets-green-light-from-us-panel-1.20137 Would you change the human germ line and eliminate deleterious disease genes or design your own child?

Stem Cells Stem cells are the progenitors of many different cell types, depending upon which type of stem cell is used (e.g., bone marrow stem cells, neural stem cells, embryonic stem cells) Stem cell therapy-the goal is to repair damaged tissue (e.g. Parkinson’s disease, spinal cord injury, heart disease) See https://www.youtube.com/watch?v=tD0HSFe3mrY